Screening for efficient sgRNAs in HEK293 cells
The fourth generation of cytosine base editor (BE4) expresses a Cas9n
(D10A) fused to cytidine deaminase (rAPOBEC1) and two copies of uracil
glycosylase inhibitor (UGI). To test whether it works in our hand, we
first transfected BE4 and pEGFP-sgRNAs in cells that stably express EGFP
(Fig. 1A) 9 (Komor et al., 2017). HEK293
cells stably expressing EGFP were generated by transfection of plasmid
pEGFP-N1. EGFP expression was checked under a fluorescence microscope.
Result showed that up to 90% of cells with fluorescence (Fig. 1D).
Positive clones (HEK293-EGFP) were selected using G418 (500μg/mL). Two
sgRNAs were designed to target EGFP gene (Fig. 1E, Supplementary Table
1). The specificity score of both sgRNA-1 and sgRNA-2 were 75%, and
83% respectively based on software analysis (https://benchling.com/).
HEK293-EGFP cells were co-transfected with BE4 and plasmids encoding
sgRNAs (pEGFP-sgRNA1 and pEGFP-sgRNA2). Two days after transfection,
EGFP fluorescence intensity was analyzed by fluorescence microscopy
(Fig. 1D). Majority of cells transfected with pEGFP-sgRNA1 still express
relatively high levels of EGFP (64%), while cells transfected with
pEGFP-sgRNA2 exhibited weak signal with an intensity of 36%, indicating
EGFP was knocked down more efficiently (Fig. 1D, F). Genomic DNA was
extracted for PCR and sequencing to confirm successful base editing.